One of the most important applications of the new recombinant DNA technology is in the production of safe vaccines against infectious diseases and the synthesis of defined proteins against which antisera can be raised for experimental, industrial and diagnostic purposes. Theoretically, these goals can be achieved by the synthesis of appropriate antigens in microorganisms such as the yeast Saccharomyces cerevisiae. The antigen would be expressed from an appropriate expression vector such as described in European Patent Application EPA2-0073635.
Correct presentation of the antigen to an animal or human immune system is a key requirement for an effective sub-unit vaccine or immunogen. Presentation has been a major problem with potential vaccines and immunogens made by recombinant DNA as well as for those based on chemically synthesized epitopes. An ideal immunogen is a polymer of multiple antigenic determinants assembled into a high molecular weight carrier. A good immunogen should also have the maximum number of epitopes exposed. These requirements can be difficult to achieve by random chemical coupling of antigens to a carrier. An ideal situation would be where the antigen was presented in the correct conformation on the surface of a large particular complex. Furthermore the particular nature of such a system would facilitate the purification of the antigen by simple physical means.
These requirements are rarely achieved by the simple synthesis of monomeric proteins by recombinant DNA technology or chemical synthesis.
Prior to the present invention, the only self-assembling, particular antigen presentation system for production of immunogens in yeast was based on the fusion of antigens (e.g. the Herpes Simplex Virus I (HSV-I) glycoprotein D or a Poliovirus antigen) to the Hepatitis B surface antigen (HBsAg) protein via recombinant DNA technology (Valenzuela et al 1985 Biotechnology 3, 323). These fusion proteins aggregate to form 22 nm particles. This system has serious disadvantages: (1) yields are very low; (2) particles do not form in the yeast cell but are a by-product of the extraction process (Hitzeman et al 1983 Nucl. Acids Res. 11,27450. This imposes limitations on the production process; (3) some fusions do not form particles; (4) the HBsAg component exerts immunodominance in some cases, i.e. antibodies are made preferentially to HBsAG.
Kniskern et al in Gene 46 135 (1986) have reported that Hepatitis B core antigen (HBcAg) forms particles in yeast at very high levels, but there has been no published report of its use to carry other epitopes. As these particles are reported to be highly immunogenic in mice they may exert immunodominance like the HBsAg particles.
A corresponding system in bacteria has been reported by Haynes et al (Bio/Technology 4 637 1986). Tobacco mosaic virus (TMV) coat protein is the entity which assembles into a particular structure. When the TMV coat protein is expressed in E. coli and then purified it is possible to assemble the proteins into polyvalent particles in vitro. Fusion proteins made by TMV coat protein and another protein may product hybrid particles that are immunogenic. A polio epitope of only eight amino acids has been tested and shown to be immunogenic although it was four times less immunogenic than the Salk vaccine. This system has the potential advantages that the size of the particle may be varied experimentally and mixed particles (i.e. carrying more than one type of epitope) could possibly be easy to produce. However, an anticipated disadvantage is that only relatively small antigens will be able to be added to the TMV particle.
Mellor et. al. in Nature 313 243 (1985) disclose a fusion protein comprising the product of a yeast Ty open reading frame ORF1 (now designated the TYA gene), an open reading frame ORF2 (now designated the TYB gene) and nucleic acid coding for interferon. However, there is no disclosure or suggestion in this paper that the fusion proteins produced, or indeed any Ty protein, are or might be capable of assembling into particles.